PROJECT SUMMARY/ABSTRACT The prevalence of allergic diseases and asthma is increasing worldwide. These diseases often occur together in the same individual, suggesting the presence of common underlying immunopathogenic factors. The long-term objective of this project is to investigate the fundamental immunological mechanisms involved in the development of allergic diseases. It is becoming increasingly clear that cytokines produced by epithelial cells at the barrier surface, including thymic stromal lymphopoietin, IL-25, and IL-33, play an important role in shaping type 2 immunity and in the pathophysiology of allergic diseases in humans. However, major gaps still remain in our knowledge concerning the molecular and cellular control of production and secretion of these cytokines. The objective of this application is to address these gaps by focusing on IL-33. IL-33 is generally stored in the nucleus of non-hematologic cells. We found recently that exposure of human airway epithelial cells to a fungal allergen, Alternaria alternata, evokes a rapid extracellular release of ATP, which triggers sustained increases in intracellular calcium concentration and IL-33 secretion into the extracellular milieu. We also found that both ATP release and IL-33 secretion are abolished when levels of reactive oxygen species (ROS) are reduced by ROS scavengers and small molecule activators of Nrf2, which increases the expression of endogenous antioxidant enzymes. Secreted IL-33 had been processed to a 19 kDa form from the full-length 31 kDa form. Therefore, we hypothesize that, upon exposure to airborne allergens, the oxidative stress response in airway epithelium triggers extracellular release of ATP, resulting in proteolytic processing and secretion of highly active IL-33. The proposed study is designed to investigate the key steps of this process in detail. In Aim 1, we will determine how oxidative stress initiates and sustains the release of ATP from airway epithelial cells, a critical step that triggers IL-33 secretion. In Aim 2, we will investigate how airborne allergen exposure induces oxidative stress in airway epithelium by studying roles for the receptor for advanced glycation endproducts (RAGE). In Aim 3, we will examine the cellular mechanisms involved in the processing and secretion of IL-33 by studying the roles for the epithelium's endogenous protease. We will employ a combination of complementary molecular, cell biological, pharmacological and immunological expertise in the laboratories of Dr. O'Grady and Dr. Kita. Novel and robust in vitro and in vivo models have been developed for this project. These studies will provide a better understanding of how the epithelium responds to environmental allergens and will define the key immunopathophysiologic mechanisms responsible for secretion of IL-33. Ultimately, these studies will provide an important characterization of key molecule(s) involved in allergen-induced immune responses, allowing for identification of novel therapeutic target(s) to treat and ideally to prevent immune-mediated diseases, such as asthma, chronic rhinosinusitis, and allergic disorders.